1. Field of the Invention
The present invention generally relates to nitroimidazole ester analogues, such as metronidazole ester analogues, and more particularly relates to novel metronidazole ester analogues that have anti-microbial activity and therapeutic applications thereof.
2. Description of Related Art
Metronidazole and similar compounds such as tinidazole have long been known as anti-microbial compounds. For example, metronidazole is known to be useful for the treatment of various conditions, including amebiasis (acute amebic dysentery), trichomoniasis, bacterial vaginosis, and Helicobacter pylori infections associated with duodenal ulcer disease.
It has been estimated that a significant number of adults are infected with H. pylon. These persons, although infected, are typically not treated until and unless they develop painful symptoms such as associated with ulcers. However, this bacteria is implicated in two different cancers.
Only a few antibiotic agents are presently known that have any effect on H. pylori, and resistance to these is developing. Metronidazole is one of these.
U.S. Pat. No. 4,160,827, issued Jul. 10, 1979, inventors Cho et al., describes phosphates of metronidazole which are water soluble and said to be useful for treating those diseases for which metronidazole is known to be useful.
Cho et al., Journal of Pharmaceutical Sciences, 74(8), pp. 883-885, 1985, disclose amino acid esters of metronidazole with improved solubility (phenylalanine ester and histidine ester). Cho et al., Journal of Pharmaceutical Sciences, 71(4), pp. 410-414, 1982, describe water soluble metronidazole phosphate.
U.S. Pat. No. 4,482,722, issued November 13, 1984, inventors Thorbek et al. discloses the N,N-dimethylglycine ester of metronidazole, which has improved solubility and is said to be useful in the systemic treatment of anaerobic infection by parenteral administration.
Johansen and Larsen, International Journal of Pharmaceutics, 26, pp. 227-241, 1985, describe hydrolytic degradation rates of certain aliphatic and aromatic esters of metronidazole. These esters are described as prodrugs with increased water solubility (such as useful in preparing parenteral dosage forms) and said to have improved transport against different biological membranes. Johansen et al., Interniational Journial of Pharmaceutics, 32, pp. 199-206, 1986, similarly describe permeation studies with a series of aliphatic ester prodrugs.
Cosar et al., xe2x80x9cNitro-imidazoles-Prxc3xa9paration et activitxc3xa9 chimio-thxc3xa9rapeurique,xe2x80x9d Arzeiniittel-Forsch, 16(1), 23-9 (Fr), 66:2512e, 1967, describe various esters of metronidazole and metronidazole analogs of which they report the most interesting are numbers 23 and 47 (of Tables 5 and 6). These are said to be active against Trichomonas vaginalis and others infectious agents. No. 47 is said to be distinguished by a weak toxicity and a good tolerance even upon prolonged administration.
Among formulations for various applications of metronidazole are those described by U.S. Pat. No. 5,840,744, issued Nov. 24, 1998, inventor Bordman, (describing a metronidazole composition that may be topically applied), and U.S. Pat. No. 6,017,516, issued Jan. 25, 2000, inventor Mody et al (describing a dental formulation including metronidazole benzoate and chlorhexydineglycanate).
Although metronidazole is one of the four antibiotics useful against bacteria, such as H. pylori, unfortunately some major problems have been encountered in the uses of metronidazole, such as in the treatment of H. pylori infections. Relapse is common and as earlier noted, various resistant strains are emerging. Accordingly, new compounds would be useful in the various therapeutic applications to which metronidazole may be put. Such new compounds could also provide efficacy against resistant strains of microbes, such as resistant H. pylori strains.
In one aspect of the present invention, new nitroimidazole compounds are provided. Novel nitroimidazoles of the present invention typically have the structures generally illustrated by Formula A. 
In Formula A, the R moiety has at least one aromatic ring. If the at least one aromatic ring is a single phenyl bonded directly to the carbonyl carbon of the ester linkage, then it is substituted with an amide substituent, or if not an amide substituent then with at least two different substituents. If the at least one aromatic ring is not bonded directly to the carbonyl carbon of the ester linkage, and is a single phenyl, then it is substituted with at least one substituent. Preferred substituents are nitro and halogen. When the substituent is amide, then it may be primary or secondary. The at least one aromatic ring may be or include a 5 or 6 membered heterocycle, or may be multiple rings formed from all carbon atoms or including one or more of the same or different heteroatoms. Heteroatoms, when present, may be oxygen, nitrogen and/or sulfur atoms.
Further in Formula A, the Rxe2x80x2 moiety may be hydrogen, halogen, hydroxy, xe2x80x94SH, an alkoxy with 1-8 carbons or where one or more sulfur atoms replaces carbon, an alkyl with 1-8 carbons or where one or more sulfur atoms replaces carbon, and where n is an integer from 2 to 8.
Particularly preferred compounds are metronidazole ester analogs wherein Rxe2x80x2 is hydrogen and n is 2, as illustrated by Formula B. 
These inventive analogs preferably have anti-microbial properties. Some of the particularly preferred embodiments have remarkable toxicity against even resistant strains of microbes, such as resistant H pylori. Five particularly preferred embodiments, the structures of which are shown below, have demonstrated extraordinarily potent anti-bactericidal activity in an assay with H pylori that is predictive of efficacy in human treatment. 
In the above-noted particularly preferred embodiments, Formula 3C is a di-ester where the at least one aromatic ring is a single phenyl bonded directly to the carbonyl carbon of the ester linkage, and is substituted with a nitro group as one substituent and with another substituent as a metronidazole ester moiety. The Formulas 1C and 2C embodiments illustrate respectively a six member heterocycle where the heteroatom is nitrogen and a five member heterocycle where the heteroatom is oxygen (and the ring is substituted with a nitro). The Formulas 4C and 5C embodiments illustrate multiple rings, where there is a phenyl substituent upon the nitrogen containing quinolyl for Formula 4C and both nitrogen and sulfur are present in the fused rings of Formula 5C.
Novel compounds of this invention are usefully formulated with pharmaceutically suitable carriers and administered for their biological activities, such as in anti-microbial applications.
We have prepared a number of novel nitroimidazole compounds of the Formula A structure, shown below, and which have demonstrated anti-microbial activity. Preferred analogues of this invention have an enhanced anti-microbial activity as to at least one microbe, with respect to metronidazole. As will be hereinafter more fully described, many of the preferred embodiments have a Minimal Bactericidal Concentration (MBC) against at least one H. pylori strain that is less than about 25 mcg/mL and the particularly preferred five embodiments have a MBC value against a H. pylori resistant strain of between about 2-5 mcg/mL (by contrast to 89 mcg/mL for metronidazole). This means that these particularly preferred five embodiments are extremely potent and are about 45 times better at killing the resistant bacteria than metronidazole itself The assay demonstrating this potency is indicative of human anti-bacterial efficacy.
Novel nitroimidazole compounds of the present invention typically have the structure generally illustrated by Formula A. 
In Formula A, the R moiety includes at least one aromatic ring. If the at least one aromatic ring is a single phenyl bonded directly to the carbonyl carbon of the ester linkage, then it is substituted with an amide substituent, or if not an amide substituent then with at least two different substituents. If the at least one aromatic ring is not bonded directly to the carbonyl carbon of the ester linkage, and is a single phenyl, then it is substituted with at least one substituent. Preferred substituents are nitro and halogen. When the substituent is amide, then it may be primary, secondary, or tertiary. The at least one aromatic ring may be a 5 or 6 membered heterocycle, or maybe multiple rings formed from all carbon atoms or including one or more of the same or different heteroatoms. Heteroatoms, when present, may be oxygen, nitrogen and/or sulfur atoms.
Further in Formula A, the Rxe2x80x2 moiety may be hydrogen, halogen, hydroxy, xe2x80x94SH, an alkoxy with 1-8 carbons or where one or more sulfur atoms replaces carbon, an alkyl with 1-8 carbons or where one or more sulfur atoms replaces carbon, and where n is an integer from 2 to 8.
Particularly preferred compounds are metronidazole ester analogs wherein both Rxe2x80x2 and Rxe2x80x3 are hydrogen and n is 2, as illustrated by Formula B. 
The remarkable anti-microbial activity that has been found for compounds of this invention, and the wide variations of structures possible as the R moiety of Formulas A and B, are quite surprising.
Some illustrative ring moieties for R are, e.g., pyridyl, furfuryl, quinolyl, phenothiazinyl, 9-oxo-9H-thioxanthenyl 10, 10-dioxide, chromonyl, anthraquinonyl, acetamidophenyl, quinolonyl, acridyl, benzimidazolyl, benzothiazolyl, benzopyranyl, benzoxazinyl, benzoxazolyl, pyrazinyl, phenazinyl, quinazolinyl, 1,2,3-thiadiazolyl, thiazinyl, tetrazolyl, thiazolyl, triazolyl, and triazinyl. Illustrative structures for various of these preferred R moieties are represented by embodiments of Formulas 1C-15 below, of which the Formulas 1C-5C compounds are particularly preferred. 
The Formula 1C and 2C embodiments illustrate inventive compounds where the at least one aromatic ring is a single heterocyclic that is bonded directly to the carbonyl carbon while in the Formula 13 embodiment the heterocyclic is further substituted with an aromatic. The Formula 3C embodiment on one hand can be viewed as a di-ester, but when looked at in the context of Formula B, has two different sub stituents (a nitro and another met ronidazole ester moiety). The Formula 8 embodiment has the at least one aromatic ring as a phenyl that is bonded directly to the carbonyl carbon with the phenyl having two different sub stituents (neither of which is an amide). The Formula 14 embodiment, however, has an amide substituent. The Formula 4C, 5C, 6, 7 and 10 embodiments illustrate novel compounds having heterocyclics as part of multiple rings. The Formulas 4C-7 and 10 embodiments also illustrate the presence of different heteroatoms in the same or different rings and a carbonyl carbon as part of the rings, whereas the Formula 15 embodiment has two fused N-containing rings. The Formulas 9 and 11 embodiments illustrate multiple fused rings where all the ring members are carbon atoms. Formula 11, as well as Formula 12, also illustrate compounds where the at least one aromatic ring of the R substituent for Formula B is not bonded directly to the carbonyl carbon of the ester linkage. When the at least one aromatic ring is a single phenyl, then it is substituted with at least one substituent, such as the preferred nitro.
The ester linkage is believed essential for the enhanced anti-microbial activity of these metronidazole analogs. The substituent linked via the ester linkage (shown as the xe2x80x9cRxe2x80x9d moiety of Formulas A and B) is believed to convey an increased lipophilic character to the inventive compounds so that they have enhanced penetration into microbial membranes. Without being limited by theory, we view the R substituent as having or conveying the character of one or more an oxidizing group (quinonyl, nitro, or the like).
The novel compounds of this invention have at least one biological activity, and preferred embodiments have the at least one biological property as an anti-microbial activity, more preferably that is improved with respect to metronidazole. Metronidazole is known to have a variety of biological activities, most importantly anti-microbial activities or properties. The term xe2x80x9cbiological activityxe2x80x9d as used herein means a property with utility in the treatment or prevention of disease or disorders affecting animals or humans, or in the regulation of an animal or human physiological condition. Among the uses known for metronidazole, to which uses compounds of this invention may be put, are in treating H. pylori infections, amebic dysentery, giardiasis, trichomonas vaginalis, and other parasitic diseases (e.g. clostridium difficile). The inventive compounds will be formulated in such therapeutic applications as suitable for the disease being treated, such as to be administered orally, topically, or by i.v.
For example, as demonstrated by the data of Table 1 in the experimental section, preferred embodiments are effective in killing H. pylori and do so with a potency a number of that is useful in treating patients having on the order of 106 to about 107 organisms in their gastric mucosa. Indeed, particularly preferred embodiments have substantially enhanced H. pylori killing effects with respect to metronidazole.
Many compounds of this invention have limited water solubility; however, drug delivery formulations are known and useful that allow materials soluble in any of water, oil, or other solutes (such as alcohol) to be formulated for delivery of drugs by any means of administration desired, such as by i.v. and i.p. Thus, for example, U.S. Pat. No. 5,629,021, issued May 13, 1997, inventor Wright, incorporated by reference, discloses micellar nanoparticles that may be formed into stable dispersions in aqueous solutions and buffers, and such nanoparticles can be used in delivering the novel compounds of this invention. Other synthetic particles suitable for administering the novel compounds, such as liposomes, nonphospholipid vesicles, and microcapsules, are also known and can be used in preparing formulations for delivering the novel metronidazole ester analogues of this invention.
Among the known formulations for delivery of biologically active agents such as metronidazole are those including oils (e.g. U.S. Pat. No. 5,143,934, issued Sep. 1, 1992) and buffering systems (e.g. U.S. Pat. No. 5,840,744, issued Nov. 24, 1998). U.S. Pat. No. 5,618,559, issued Apr. 8, 1997 discloses pharmaceutical composition with a modified-release profile for daily dosing of metronidazole.
Thus, the inventive analogues of the present invention can be prepared in formulations analogous to these various known teachings for metronidazole formulations and then be used to treat a patient, such as to treat an infection caused by a microorganism by administering a composition including an effective amount of the inventive analogue to the patient. Again, administration can be as appropriate, such as oral, topical, nasal or into the blood stream.
For one example, compositions of the present invention may be formulated as granules for oral administration which include the metronidazole ester analogues, together with various well-known tableting agents, excipients, and the like. Such tablets are contemplated for treating patients infected with a Helicobacter species, such as Helicobacter pylori, particularly in treating resistant species.
Alternatively, as another example, compositions of the present invention may be formulated where the novel compounds are encapsulated in synthetic particles such as liposomes, micellular nanoparticles, or nonphospholipid vesicles. Such formulations can be administered orally for treating patients infected with a Helicobacter species. Novel compounds of this invention may be incorporated into such particles, particularly for treating resistant H. pylori. 
The clinical resolution of an infection or disease may be readily determined by a clinician of ordinary skill in the art, such as by microbiological testing or disappearance of clinically characteristic symptoms. Thus, the particular form of administration and the dosage of nitroimidazoles in accordance with this invention and/or the length of treatment may be increased or decreased based on the type of infection, the degree of susceptibility of the microorganism, the age and general health of the patient, and like factors of which a clinician of ordinary skill in the art is aware.
Further, with respect to H. pylori, present clinical practice frequently includes administering an anti-microbial in combination with other active ingredients, such as bismuth and amoxicillin, bismuth and tetracycline, and others. For another example, U.S. Pat. No. 5,618,840, issued Apr. 8, 1997, inventor Wright, describes an antibacterial oil-in-water emulsion that inhibits the growth of H. pylori, which emulsions can be administered to individuals, for example orally.
Example A gives two illustrative (prophetic) preparations of Formula A compounds. Further, compounds of this invention of Formula B may be prepared by various methods, such as by (a) the acylation of metronidazole with the corresponding organic acid chloride, or (b) the reaction of two substituents with a coupling reagent (such as DCC, EDCI, DIPC, and so on) and a catalytic agent (such as DMPA).